Species differences in the distribution of adrenal free and sulfoconjugated catecholamines

Summary We determined free and sulfoconjugated catecholamines (CA) in adrenals of several species by reverse-phase high performance liquid chromatography (HPLC) with electrochemical detection. The two main adrenal CA, free epinephrine (E) and norepinephrine (NE) from eight species (guinea-pig, rat, dog, mice, bovine, cat, green-monkey and human) were considerably different both in the total amount as well as their relative proportions. Free dopamine (DA) content also differed from species to species but this CA was present in a relatively constant proportion, representing about 1% of the total free CA. Phenolsulfotransferase (PST) activity was present in all of the adrenals. Sulfoconjugated CA, however, were only selectively present: E and NE sulfate were entirely absent but in most of these species DA sulfate was detected in a proportion corresponding to 1–10% of the total (free + sulfoconjugated) DA. The adrenal DA sulfate concentrations did not parallel the adrenal PST activity, indicating that this enzyme can not be considered to be an index of the CA sulfates present in this organ.Abbreviations CA catecholamines - DA dopamine - DHBA dihydroxybenzylamine - E epinephrine - HPLC high performance liquid chromatography - NE norepinephrine - PST phenolsulfotransferase     

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: experimental and clinical studies.

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.     

Plasma free and sulfoconjugated catecholamine responses to varying exercise intensity

Plasma free catecholamines rise during exercise, but sulfoconjugated catecholamines reportedly fall. This study examined the relationship between exercise intensity and circulating levels of sulfoconjugated norepinephrine, epinephrine, and dopamine. Seven exercise-trained men biked at approximately 30, 60, and 90% of their individual maximal oxygen consumption (VO2max) for 8 min. The 90% VO2max period resulted in significantly increased plasma free norepinephrine (rest, 219 +/- 85; exercise, 2,738 +/- 1,149 pg/ml; P less than or equal to 0.01) and epinephrine (rest, 49 +/- 49; exercise, 555 +/- 516 pg/ml; P less than or equal to 0.05). These changes were accompanied by consistent increases in sulfoconjugated norepinephrine at both the 60% (rest, 852 +/- 292; exercise, 1,431 +/- 639; P less than or equal to 0.05) and 90% (rest, 859 +/- 311; exercise, 2,223 +/- 1,015; P less than or equal to 0.05) VO2max periods. Plasma sulfoconjugated epinephrine and dopamine displayed erratic changes at the three exercise intensities. These findings suggest that sulfoconjugated norepinephrine rises during high-intensity exercise.     

Plasma free and sulfoconjugated catecholamines during sustained exercise

Previous research established a relationship between circulating sulfoconjugated norepinephrine (NE-SO4) and oxygen consumption at various exercise intensities. In this study, the stability of the NE-SO4 response was examined during sustained exercise at a constant relative intensity. Seven trained men bicycled at 78 +/- 3% of their maximal O2 consumption for 28 min and then rested on the ergometer for a comparable duration. After a 30-min rest, plasma samples were collected through an indwelling catheter at 7-min intervals during the exercise and recovery periods. Free NE and epinephrine increased sixfold during exercise. These changes were accompanied by increases in sulfoconjugated catecholamines, but only NE-SO4 achieved statistical significance (rest, 712 +/- 602; exercise, 1,329 +/- 1,163 pg/ml). This occurred at three collection periods (14, 21, and 28 min). Approximately 35, 52, and 95% of NE, epinephrine, and dopamine, respectively, existed as sulfoconjugated during exercise. Subject variation was present in the sulfoconjugated catecholamine response that could not be attributed to corresponding differences in circulating free catecholamine release. These findings implicate blood flow as a factor in the sulfoconjugation of NE, but not epinephrine or dopamine.     

Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100mM boric acid buffer containing 10mM SDS and 10mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0 x 10(-7) to 9.0 x 10(-7) M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.     

Is the platelet phenolsulfotransferase involved in the sulfoconjugation of plasma catecholamines?

Catecholamines are predominantly present in the sulfoconjugated forms in human plasma. Phenolsulfotransferase (EC 2.8.2.1), which catalyses the sulfation of phenolic compounds, is widely distributed in human tissues. In blood, a phenolsulfotransferase, more specific for catecholamine sulfation is found exclusively in platelets. Free and sulfoconjugated catecholamines were measured in plasma and platelets of healthy volunteers and compared with those present in patients with uremia or pheochromocytoma to determine the ability of platelet phenolsulfotransferase to sulfurylate plasma catecholamines. In patients with pheochromocytoma, the rise in free and sulfoconjugated plasma catecholamines is accompanied by a simultaneous rise of these molecules in platelets. In uremia, where the level of plasma catecholamines is normal, the rise in the sulfoconjugates is not accompanied by a concomitant increase in either free or sulfoconjugated catecholamines in platelets. Platelet phenolsulfotransferase activity remains unchanged in pheochromocytoma and uremia. These data indicate that the platelet phenolsulfotransferase is involved in the sulfation of the catecholamines present in platelets, but its contribution, if any, to the high level of sulfoconjugated catecholamines found in plasma is negligible. This assertion is confirmed by our observations in thrombocytopenic patients. Indeed, despite the very low number of platelets and the absence of plasma phenolsulfotransferase activity, thrombocytopenic patients have normal plasma levels of free and sulfoconjugated catecholamines.     

Assay of free and conjugated catecholamines by high-performance liquid chromatography with electrochemical detection

A rapid and simple method for the analysis of free and conjugated catecholamines in body tissues and fluids is described. The free catecholamines were isolated by standard alumina procedures before and after hydrolysis of the conjugated compounds to free compounds by heating the samples in perchloric acid. Free catecholamines were then separated by high-performance liquid chromatography and detected by electrochemical detection. Conjugated compound was the difference between the total and free amount in each sample. This method was utilized to measure free and conjugated norepinephrine, epinephrine, and dopamine in human urine and rat adrenal gland, and to measure free and conjugated dopamine in rat whole brain and kidney.     

Application of high-performance liquid chromatography with a chemiluminescence detection system to determine catecholamines in urine

Chemiluminescence generated with the reaction of bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide was applied to a detection system for high-performance liquid chromatography to determine fluorescamine-labeled catecholamines. The sensitivity of the chemiluminescence detection system with 25 fmol of detection limit was approximately 20 times higher than that of a conventional fluorescence detection system. Norepinephrine and dopamine in human urine were determined by the use of the new high-performance liquid chromatography detection system with the coefficient of variation of less than 4.0%. Good correlations (r = 0.998 for norepinephrine and r = 0.999 for dopamine) were obtained between the values by the present method and the conventional method.     

Methanol plug assisted sweeping-micellar electrokinetic chromatography for the determination of dopamine in urine by violet light emitting diode-induced fluorescence detection

The use and limitations of a methanol plug assisted sweeping-micellar electrokinetic chromatography (sweeping-MEKC) method is described. Using naphthalene-2,3-dicarboxaldehyde (NDA)-labeled dopamine as a model compound, this new method was also used in the determination of dopamine in actual urine samples. An inexpensive violet light emitting diode (LED) was used for the light source, because this is suitable for fluorescence excitation. The number of theoretical plates of the analyte was determined to be approximately 1 x 10(5) and approximately 2 x 10(5) by means of MEKC and sweeping-MEKC and this was improved to approximately 1 x 10(6) when the methanol plug assisted mode was applied. In addition, the detection limit of NDA-labeled dopamine was determined to be 9.1 x 10(-7) and 1.2 x 10(-8)M by means of MEKC and sweeping-MEKC and this was improved to 4.7 x 10(-9)M when the methanol plug assisted sweeping-MEKC mode was applied.     

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.     

Isomer specific kinetics of dopamine beta-hydroxylase and arylsulfatase towards catecholamine sulfates

Both isomers of epinephrine sulfate were synthesized, unequivocally identified by 1H-NMR and highly purified from catecholamines (less than 90 ppm). Bacterial as well as pig liver arylsulfatase A and B demonstrated a higher substrate turnover of epinephrine-4-sulfate, norepinephrine-4-sulfate and dopamine-4-sulfate as compared to the 3-sulfate isomers. The arylsulfatase B however, is less important for the deconjugation of these sulfoconjugates than arylsulfatase A. Since arylsulfatase A occurs in most human tissues, it might be of physiological significance in the deconjugation of the catecholamine sulfate isomers. Furthermore the kinetic data at pH 7.4 and 6.9 suggest the increased cleavage of the sulfate group, e.g. during exercise-induced acidosis. In contrast to results reported in the literature, dopamine sulfates were no substrates of dopamine beta-hydroxylase.     

Assay of norepinephrine and dopamine in the rat brain after microwave irradiation

Rapid inactivation of enzymes prior to the assay of rat brain catecholamines was evaluated. Regional levels of norepinephrine and dopamine were measured by high performance liquid chromatography with electrochemical detection after enzyme inactivation by microwave irradiation at levels of 1.3 kw and 5 kw, and compared with decapitation. The differences found in regional levels of catecholamines between the two methods of euthanasia indicate that rapid inactivation of brain enzymes is necessary for accurate analysis of catecholamines in rat brain.     

A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrochemical detection

Simple and sensitive methods for the determination of plasma catecholamines are of great interest since the level of catecholamines in plasma reflects the activity of the sympatho-adrenal system. In the present work a previously described procedure based on high pressure liquid chromatography with electrochemical detection has been adapted for assay of plasma catecholamines. This method permits simultaneous detection of noradrenaline, adrenaline and dopamine in concentrations down to 0.1 nmol/1 in less than one ml plasma.     

Analysis of catecholamines and related substances using porous graphitic carbon as separation media in liquid chromatography-tandem mass spectrometry

Capillary porous graphitic carbon (PGC) columns have been utilized for separation of several catecholamines and related compounds (i.e. L-tyrosine, L-DOPA, 3-O-methyl-DOPA, dopamine, 3,4-dihydroxy-phenyl-acetic acid (DOPAC), homovanillic acid, noradrenaline, vanillomandelic acid and adrenaline) on-line with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of a mobile phase without ion-pairing agents and with high content of organic modifier facilitated the coupling to the selective and sensitive mass spectrometric detection. Minimum detectable sample concentration (MDC sample) for noradrenaline, dopamine and L-tyrosine in a standard solution was estimated to 3, 10 and 30 nM, respectively (3 S/N corresponds to MDQ for L-tyrosine of approximately 8 x 10(-14)mol). The developed strategy was applied for analysis of brain tissue, i.e. a substantia nigra (ns) sample.     

Dopamine and norepinephrine in the alimentary tract changes after chemical sympathectomy and surgical vagotomy

The aim of this study was to examine the distribution of dopamine and norepinephrine in the proximal alimentary tract of the rat and to assess the contributions of sympathetic and vagal fibers to the tissue concentrations of both catecholamines. Tissues were extracted in perchloric acid and the catecholamines were separated by high pressure liquid chromatography and detected electrochemically. In untreated rats (controls) both catecholamines were concentrated in the gastric muscle but norepinephrine levels were 6-8 times higher (corpus, dopamine 35 +/- 7 ng . g-1, norepinephrine 265 +/- 50 ng . g-1, mean +/- SE, n = 6). In the mucosa norepinephrine concentrations were 10-12 times higher (corpus, dopamine 12 +/- 3 ng . g-1, norepinephrine 140 +/- 26 ng . g-1). Chemical sympathectomy (6 hydroxydopamine, 100 mg . kg-1 ip 3 days) significantly reduced dopamine concentrations in muscle and norepinephrine in muscle, mucosa, pylorus and duodenum. In all tissues the effects on norepinephrine were greater. Surgical vagotomy significantly reduced dopamine concentrations in the gastric muscle, but not the mucosa. Norepinephrine concentrations in the stomach of vagotomized rats were significantly reduced only in the pylorus. Differences in the relative concentrations of dopamine and norepinephrine in gastric tissues of the normal rat and differences in the effects of sympathectomy and vagotomy suggest that dopamine and norepinephrine exist, to an extent, in separate populations of cells and that dopamine is not merely a precursor of norepinephrine. Gastric mucosal dopamine, which was mainly unaffected by either treatment, may exist in APUD cells.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Regional changes in catecholamine content of the pregnant uterus

High-pressure liquid chromatography with electrochemical detection was used to identify and measure catecholamines in rat, rabbit, sheep, guinea-pig and human uteri and follow changes with pregnancy. Noradrenaline was consistently the major catecholamine and pregnancy caused a regionally specific fall in its concentration which, in rat, rabbit and guinea-pig, was associated with a decline in total content. Adrenaline was undetectable (less than 10 pmol/g myometrium) in all species and at all gestational ages studied. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected at high concentrations in guinea-pig and particularly sheep uterus. In guinea-pig uterus the dopamine/DOPAC ratio fell dramatically with pregnancy, suggesting that increased quantities of dopamine were released and catabolized. The dopamine/noradrenaline ratios suggested that dopamine is stored with noradrenaline in adrenergic neurones in guinea-pig myometrium and within an additional neuronal or cellular store(s) in sheep uterus.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Flow-injection chemiluminescence determination of catecholamines based on their enhancing effects on the luminol-potassium periodate system.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis is described for the determination of four catecholamines, dopamine, adrenaline, isoprenaline and noradrenaline, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. The optimized chemical conditions for the chemiluminescence reaction were 1.0 x 10(-4) mol/L luminol and 1.0 x 10(-5) mol/L potassium periodate in 0.2 mol/L sodium hydroxide (NaOH). Under the optimized conditions, the calibration graphs relating the CL signal intensity (peak height) to the concentration of the analytes were curvilinear and they were suitable for determining dopamine, adrenaline, isoprenaline, and noradrenaline in the range 0.1-10 ng/mL, 0.1-100 ng/mL, 1-100 ng/mL and 5-50 ng/mL, respectively, with the relative standard deviations of 0.8-1.7%. The detection limits of the method are 0.02 ng/mL for dopamine, 0.01 ng/mL for adrenaline, 0.1 ng/mL for isoprenaline and 2.0 ng/mL for noradrenaline. The sampling frequency was calculated to be about 60/h. The selectivity of the method was good, because a series of common ions or excipients, such as K(+), Ba(2+), CO(3)(2-), NO(3)(-), SO(4)(2-), PO(4)(3-), sodium citrate, sodium bisulphite, oxidate dopamine, starch, lactose, carbamide and gelatin, could not produce interference when their concentrations were 1000-fold than those of dopamine. The present method was successfully applied to the determination of the four catecholamines in pharmaceutical injections.     

Inhibitory actions of dopamine on Limulus visceral muscle involve a cyclic AMP-dependent mechanism

1. The catecholamines dopamine, epinephrine and norepinephrine were detected in alumina extracts of Limulus midgut tissue using high performance liquid chromatography with electrochemical detection. Moderate levels of norepinephrine (28.2 +/- 2.1 ng/g) and dopamine (24.0 +/- 5.2 ng/g) were detected in the midgut, while epinephrine levels (7.4 +/- 0.9 ng/g) were less. Catecholamines were present in all regions along the longitudinal axis of the midgut, and norepinephrine and dopamine levels were highest in posterior regions. 2. Catecholamines decreased muscle tonus and inhibited spontaneous contractions of the Limulus midgut. Dopamine typically decreased spontaneous midgut activity at doses of 10(-8) M or greater, and produced inhibitory actions on all regions of the Limulus midgut. In some preparations epinephrine and norepinephrine elicited a secondary rhythmicity. The actions of dopamine opposed the excitatory effects produced by either proctolin or octopamine. 3. Catecholamines significantly elevated levels of cyclic AMP in Limulus midgut muscle rings. Dopamine (10(-5) M) increased cyclic AMP with a time course consistent with its physiological effects. Forskolin and several methyl xanthines increased Limulus midgut cyclic AMP levels and mimicked the inhibitory effects of dopamine on the isolated midgut preparation. Cyclic nucleotide analogues also produced dopamine-like effects on the isolated midgut preparation. Inhibition of cyclic nucleotide phosphodiesterase prior to addition of dopamine enhanced the effect of this amine to decrease baseline muscle tension. 4. The inhibitory effects of 10(-5) M dopamine on the midgut persisted in solutions of zero sodium and in the presence of tetrodotoxin. Zero calcium solutions gradually reduced spontaneous midgut activity and the effects of dopamine. Calcium channel blockers did not prohibit dopamine-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)